DESCRIPTION: This is a request for continuation of Dr. Condeelis' work on the actin binding proteins in Dictyostelium. It is focussed on the previously established intriguing finding from his laboratory, that one of the F-actin binding and cross-linking proteins in Dictyostelium, ABP-50, is elongation factor 1alpha (EF1a ). Surprisingly, EF1a is present at 1-2% of the total protein and in 7-13 molar excess over other components of the protein synthesis complex, supporting the idea of a dual role in the cell. The goal of the proposed work is to understand the interaction between EF1a, actin and the other components of the protein synthesis machinery, aatRNA, mRNA and ribosomes. The approach includes both functional and structural studies. The specific aims are to determine which of the four polypeptide elongation reactions in which EF1a is involved are affected by actin binding, to map further the actin binding sites of EF1a, to prepare mutant forms of EF1a which lack actin binding activity and to use them to study the importance of this interaction, both in vitro and in vivo, and to use blocking antibodies and cells expressing the mutant forms to test models for the interaction of the actin and aatRNA binding sites on EF1a. On the one hand, he will explore the function of EF1a in regulating the actin cytoskeleton, and on the other, the importance of actin binding as affecting mRNA localization and translation. Finally, he and his collaborators will study the structure of EF1a -actin complexes and attempt to obtain crystals of EF1a for 3D structure solution. The PI's laboratory has previously shown that bundling of F-actin by EF1a was pH sensitive in the physiological range, with bundling activity dropping sharply just above pH 7 at pH levels found in stimulated cells, including Dictyostelium. In the recent grant period, using truncated fragments of GST fusion proteins his group has shown that there are two F-actin binding domains in EF1a , one in domain I, which is sensitive to pH and no longer binds F-actin even at pH 6.8 and one in domain III which is less sensitive, while domain II does not bind to actin. Using the 3-D structure of the corresponding bacterial elongation factor EF-Tu (which has been crystallized) as a guide, I and III lie at opposite sides of the molecule. The pH sensitivity of domain I seems highly relevant to the pH dependence of bundling by the entire molecule, while the much less sensitive binding by domain III could explain the fact that EF1a continues to bind to filaments at higher pH. Preliminary studies of the interaction of the ternary complex of EF1a with aa-tRNA are presented which show that the binding of the complex to F-actin stabilizes the GTP, or active form of the factor by decreasing its off rates. Thus, F-actin is not a GEF for the factor, but the GTP form will tend to accumulate on actin filaments- there is a difference in Kd for GTP over GDP of about 3 fold. Furthermore, the binding of GTP-EF1a to aatRNA inhibits the ability of the factor to bundle F-actin shown by its shift from low speed pellet to high speed pellet.